Telomeres are the protein-DNA complexes that protect the ends of linear eukaryotic chromosomes from degradation, prevent end-to-end fusions and partake in chromosome localization and segregation (Cooper, Curr Opin Genet Dev 10: 169-77, 2000; McEachern et al., Annu Rev Genet 34: 331-358, 2000; Price, Curr Opin Genet Dev 9: 218-24, 1999). Telomere length, 15-20 kb in human embryonic or germ line cells, is maintained in part by the enzyme telomerase. In the absence of telomerase activity, about 50-200 bases of DNA are not replicated with each round of cell division, resulting in the eventual diminution in telomere size to typically 5-7 kb. At that length, cells enter a state of arrested growth called replicative senescence. The maintenance of telomere length thus is believed to play a key role in the ability of cells to avoid replicative senescence and to propagate indefinitely, as is the case with stem cells. Likewise, aberrant maintenance of telomere length is believed to underlie indefinite cellular proliferation characteristic of cancer cells (Bodnar et al., Science 279: 349-352, 1998; Bryan et al., 1997; McEachern et al., 2000).
Telomeres consist of repeating units of GC-rich DNA and terminate in a single stranded extension of the 3xe2x80x2 strand. Oxytricha nova telomeres, for example, consist of tandem repeats of (TTTTGGGG) and end with a 16 nucleotide overhang of the G-rich strand. By contrast, human telomeres have a repeating sequence (TTAGGG)n and end with a 50-100 nucleotide overhang of the G-rich strand. McEachern et al., 2000.
A number of proteins have been identified that specifically interact with the double-stranded portion of the telomere or the single-stranded 3xe2x80x2 extension at its very end. Among the most well characterized are the telomere end-binding proteins from hypotrichous ciliated protozoa (Gottschling et al., Cell 47: 195-205, 1986; Price et al., Genes Dev 1: 783-93, 1987). The xcex1 and xcex2 subunit of the O. nova Telomere End-Binding Protein (TEBP) bind specifically to the 16 nucleotide single-stranded extension at the ends of macronuclear chromosomes (Gray et al., Cell 67: 807-14, 1991) and form a ternary complex whose structure has been determined using X-ray crystallography (Horvath et al., Cell 95: 963-974, 1998). Although both protein subunits directly interact with DNA in the ternary complex, only xcex1 binds telomeric DNA by itself (Fang et al., Genes Dev 7: 870-82, 1993). The DNA binding domain in the a subunit has been mapped to the N-terminal two-thirds of the polypeptide (Fang et al., 1993) and is comprised of two xe2x80x9cOB foldsxe2x80x9d (Horvath et al., 1998). In vitro reconstituted xcex1-DNA complexes are substrates for telomerase, whereas xcex1-xcex2-DNA complexes are not; an observation which may indicate a function in the regulation of telomere length (Froelich-Ammon et al., Genes Dev 12: 1504-14, 1998).
The protrusion of the G-rich strand as a single-stranded overhang is conserved between ciliates (Klobutcher et al., Proc Natl Acad Sci USA 78: 3015-19, 1981), yeast (Wellinger et al., Cell 72: 51-60, 1993) and mammalian cells (Makarov et al., Cell 88: 657-66, 1997; McElligott et al., Embo J 16: 3705-14, 1997; Wright et al., Genes Dev 11: 2801-09, 1997), suggesting the existence of similar functional mechanisms in telomere maintenance. However, proteins sharing sequence homology with ciliate TEBPs were not identified in the complete S. cerevisiae genome or among the proteins that bind single-stranded telomeric DNA in vitro. Similarly, the S. cerevisiae single-stranded telomeric DNA-binding protein cdc13p has not been proposed to be homologous to the ciliate TEBPs, nor have cdc13p homologues been identified in distantly related species. (Ishikawa et al., Mol Cell Biol 13: 4301-10, 1993; Lin et al., Proc Natl Acad Sci USA 93: 13760-65, 1996; McKay et al., Nucleic Acids Res 20: 6461-64, 1992; Nugent et al., Science 274: 249-52, 1996; Virta-Pearlman et al., Genes Dev 10: 3094-104, 1996).
The apparent absence of specific end-capping proteins in some eukaryotes has been explained by the adoption of a telomere structure distinct from that found in the macronuclei of hypotrichous ciliates. This telomere structure, found at the ends of mammalian and O. fallax chromosomes, is a large duplex loop, or xe2x80x9ct loop,xe2x80x9d created by the sequestration of the single-strand overhang within the double-stranded portion of the telomeric tract (Griffith et al., Cell 97: 503-14, 1999; Murti et al., Proc Natl Acad Sci USA 96: 14436-39, 1999). In mammals, this architecture is believed to be maintained by a number of proteins, including the TTAGGG-binding factors, TRF1 and TRF2. TRF2 is believed to catalyze the sequestration of the single-stranded DNA into the duplex region of the DNA. Consistent with this notion is the observation that TRF2 can cause telomeric DNA to form t loops in vitro (Griffith et al., 1999). Other proteins have been implicated in telomere architecture and regulation, including TIN2, which was identified by its ability to interact with TRF1 (Kim et al., 1999).
The ability to manipulate telomere structure and metabolism depends on the identification of those components required for the regulation of telomere structure. Evidence has accumulated that telomerase activity itself is not determinative of telomere elongation or replication. For example, some cancer cell lines maintain telomeres in the absence of telomerase activity (Bryan et al., 1997). There is thus a pressing need in the art to identify the functional components that regulate telomere metabolism, to identify compounds that can be used to control the entry, avoidance, or exit of a cell from a state of replicative senescence. Such compounds may be useful alternatively in allowing the indefinite propagation of useful cell lines or in halting the growth of cancer cells in vivo for therapeutic purposes.
The present invention addresses this need by providing a protein that caps the very ends of human chromosomes, and a related protein that caps the ends of chromosomes in fission yeast (Schizosaccharomyces pombe). The protein of the invention is termed xe2x80x9cProtection of Telomere-1,xe2x80x9d or xe2x80x9cPot1p,xe2x80x9d or xe2x80x9cPot1 protein.xe2x80x9d Specific embodiments of these proteins are those isolated from humans and fission yeast, hpot1p and SpPot1p, respectively. Polynucleotides encoding a Pot1 protein are also provided.
The inventors have found that Pot1p binds single-stranded telomeric DNA, which is a unforeseen finding, given the apparent absence of end-capping proteins in some eukaryotes. Pot1p both stabilizes chromosome ends and regulates telomerase activity. Accordingly, compounds that stabilize or disrupt the Pot1p-DNA interaction will be useful in regulating the telomere length of a target cell or cell population. The invention thus provides a means of altering cellular life-span, for the purpose of either prolonging the life-span of useful cell populations or making cancer cells enter replicative quiescence. Useful compounds with these properties can be identified through screening methods made possible by the discovery that a Pot1 protein binds single-stranded telomeric DNA. The identification of a Pot1 protein and its encoding DNA also provides a means of developing tools to diagnose illnesses such as cancer that may involve altered expression or structure of a Pot1 protein or gene. Such tools include polynucleotide hybridization probes and antibodies specific for a Pot1 protein.
Accordingly, the invention provides isolated Pot1 proteins having the sequence set forth in SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:9, or SEQ ID NO: 11. Variants of these proteins are capable of binding single-stranded telomeric DNA and have at least 85% sequence identity with, or differ by no more than about 20 single amino acid substitutions, deletions or insertions from, a sequence set forth in SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:9, or SEQ ID NO:11. The invention also provides an isolated, naturally occurring, variant of a protein having the sequence set forth in SEQ ID NO: 13 or in SEQ ID NO:9, which may be a splicing variant. Fragments of the Pot1 proteins of the invention are capable of binding single-stranded telomeric DNA, and comprise the polypeptide having the sequence set forth in SEQ ID NO:5 or SEQ ID NO:6.
The invention further provides an isolated non-genomic polynucleotide encoding one of the aforementioned proteins. A vector comprising such a polynucleotide and a host cell comprising the vector also are provided. The polynucleotide may be included in a pharmaceutical composition, along a pharmacologically acceptable excipient, diluent, or carrier. A method of detecting or measuring the presence of a POT1 polynucleotide comprises contacting the a POT1 polynucleotide, or its complement, with a biological sample from an individual.
An antibody, or a fragment or variant thereof, is provided, which is capable of binding a Pot1 protein. A method of raising the antibody comprises isolating the antibody from an animal or isolating an antibody-producing cell from an animal, following administration of a Pot1 protein, or an antigenic fragment thereof, to the animal. An antibody of the invention may be useful in detecting or measuring the presence of a Pot1 polypeptide in an individual, by contacting the antibody with a biological sample from an individual.
The invention provides a method of increasing the life-span of a cell, by inserting a vector comprising a POT1 polynucleotide into the cell, where the POT1 polynucleotide is operably linked to a promoter that allows the polynucleotide to be transcribed. The vector comprising a POT1 polynucleotide may be administered to an individual in a pharmaceutical composition, comprising the polynucleotide and a pharmacologically acceptable excipient, diluent, or carrier. In one embodiment, the carrier is capable of preferentially delivering the polynucleotide to a specific cell population. In another embodiment, the vector comprising the POT1 polynucleotide is inserted into the cell in vitro, which then may be subsequently administered to an individual. The target cell may express a second polynucleotide that encodes an exogenous protein, such as a therapeutically useful protein.
A method of identifying a compound that interferes with the binding of a Pot1 polypeptide to single-stranded telomeric DNA comprises determining whether the candidate compound decreases the binding of the Pot1 polypeptide to a single-stranded telomeric DNA molecule in a mixture of the single-stranded telomeric DNA molecule, the polypeptide, and the candidate compound. The compound identified by this method may be formulated in a pharmaceutical composition.
A method of decreasing the life-span of a cell comprises reducing the level of Pot1p activity in a cell. The cell may be an immortal cell line, such as a cancer cell. In one embodiment, the method comprises delivering one of the compounds that interferes with the binding of a Pot1 polypeptide to single-stranded telomeric DNA.